Comet assay, a possible screening assay to classify subgroups of individuals with different radiosensitivity using high throughput scanning system for multiple samples of human blood lymphocytes.

This research was designed to identify the correlation between clinical radiosensitivity among breast cancer patients and in vitro radiosensitivity measured by alkaline comet assay in high throughput fashion. In 68 patients with breast cancer, acute adverse effects on skin after radiotherapy were clinically scored. Among these patients, maximum score was grade 0 for 10 patients, grade 1 for 28 patients, grade 2 for 25 patients, and grade 3 for 5 patients. Correlation between initial damage and skin reaction was found in breast cancer patients with grade 1 and grade 2, 3 (p= 0.034). There was also correlation between residual damage (RD) and skin reaction in these two groups (p= 0.033). Large inter-individual variation of RD was also noticed among breast cancer patients with grade 0 (17.5+- 12.7 %), or grade 2, 3 (16.8+- 10.7 %). By introducing a new analyzer with a motorinzed scanning stage for 8 slides, throughput of the comet assay data was highly improved. There was no difference in any comet parameter between cancer patients and age-matched 41 healthy volunteers. Our data suggests that the comet assay might be one of supportive assays to classify subpopulation of patients who have different radiosensitivity from normal responders with a fair discriminating capacity.International Comet Assay Workshop No.

Analysis of in vitro Radiosensitivity of mouse peripheral leukocytes by the Multi-Lane Comet Assay

16th INTERNATIONAL MOUSE GENOME CONFARENC

Strain-dependent differences in locomotor activity after local brain irradiation with 30 GyE of carbon ions

Search for genetic polymorphisms that associate with radiation-induced normal tissue injury on cancer patients\nMayumi Iwakawa and Takashi ImaiRadGenomics Project, Frontier Research CenterNational Institute of Radiological Sciences, Japan\nIntroduction Cancer patients vary considerably in normal tissue reactions after radiotherapy. Several observations have indicated that certain genetic factors play important roles in this variability. It has been hypothesized that the clinical radiosensitivity of normal tissues should be regarded as a so-called complex trait dependent on the cumulative effect of many minor genetic determinants. Thus single nucleotide polymorphisms (SNPs) on certain genes may somehow associate with the severity of normal tissue reactions after radiotherapy. It is important to uncover molecular basis underlying radiation sensitivity of normal tissues for further investigation of more complex character of cancer cells. In this study we have aimed to search for polymorphisms that associate with normal tissue radiation sensitivity of various cancer patients to open a way for achieving individual-oriented radiotherapy with high-therapeutic ratio.\nResearch strategy Our strategy is a candidate gene approach selected through the experiments using in vitro cultured human cell lines and animal models. SNPs on the selected genes have been typed using the DNA from white blood cells of cancer patients with clinical information.\nPatients and Methods1. Patients The 1071 patients in this study consist 489 breast cancer patients, 149 ovarian cancer patients, 126 prostate cancer patients, 133 head and neck cancer patients. They were registered between 2001 and 2004. Normal tissue reactions until the 3rd month after completion of the treatment were graded according to the National Cancer Institute-Common Toxicity Criteria (NCI/CTC). Late effects on normal tissues were graded according to the Radiation Therapy Oncology Group/ the European Organization for Research and Treatment of Cancer (RTOG/EORTC) scoring system and the Late Effects of Normal Tissues-Subjective, Objective, Management and Analytic (LENT-SOMA) scoring system. Patients were divided into two groups (radiosensitive and radioresistant) according to the grades determined by the above scoring systems.\n2. Candidate gene selection We have defined following three criteria to select candidate genes. (1) Genes whose expression profile showed statistically significant association with cellular radiation sensitivity. (2) Genes whose expression were induced or reduced after ionizing radiation treatment. (3) Genes whose involvement in the radiation sensitivity had been evaluated in some literatures. We have measured radiosensitivity of 32 different cultured human cancer cell lines and analyzed their gene expression profile by microarray technique. In addition, we have analyzed in vivo gene expression profile of mouse strains with different radiation sensitivity. As a total we have selected 108 candidate genes that met at least one of the above criteria.\n3. SNPs typing of the candidate genes and statistical analysis The information about SNPs on the candidate genes was obtained from JSNP database (http://snp.ims.u-tokyo.ac.jp/) and dbSNP database (http://www. ncbi.nlm.nih.gov/SNP/). Typing of the SNPs was performed by the allele-specific termination of primer extension method using a MALDI-TOF Mass spectrometer. \nResultsSix hundreds and forty three SNPs were typed for the 108 candidate genes of 346 individuals consisting of 218 breast cancer patients, 57 ovarian cancer patients and 71 prostate cancer patients. Statistical analysis of association between the SNP types and radiation sensitivity of patients has been done using SNPalyze software. So far, we found more than 12 genes that showed statistically significant association.\nConclusion This study implies that analysis of multiple SNPs on adequately selected candidate genes might be specifically suitable for identification of genetic constraints of radiation sensitivity. At present state our findings are still preliminary and require assignment of functional influence of the SNPs to the expression of gene activity that relate to the radiation sensitivity. Our study should encourage further comprehensive search for genetic polymorphisms that associate with radiation-induced normal tissue injury on cancer patients\nAcknowledgement We deeply appreciate patients who accepted to participate in our study and collaborators who participated in obtaining informed consent from medical information and providing patient\u27s blood sample. Strain-dependent differences in locomotor activity after local brain irradiation with 30 GyE of carbon ions\nMayumi Iwakawa1, Nobuhiko Takai2, Miyako Goto1, Koichi Ando2, and Takashi Imai1\nAbstract\nIn this study, mice of inbred, A/J, C57BL/6J and C3H/HeMs mice were used to reveal inter-strain differences of radiosensitivity after local brain irradiation and spontaneous locomotor activity was examined as a parameter of brain function. The whole brain of the A/J, C57BL/6J and C3H/HeMs male mice was irradiated with carbon ion beams. We measured the locomotor activity determined by the Supermex system using detection of the body heat of an animal. The daily locomotor activities of non-irradiated and irradiated A/J, C57BL/6J and C3H/HeMs mice differed significantly. This inter-strain variance became obvious immediately after irradiation, as the activity of A/J mice diminished, whereas the C57BL/6J mice became hyperactive. Daily total spontaneous locomotor activity was suppressed one day after irradiation and increased thereafter among three strains. When we also measured the locomotor activity after the dopamin receptor agonist, apomorphine, treatment, a significant increase of activity in C57BL/6J mice was detected, whereas a significant decrease in C3H/HeMs or A/J mice. In conclusion, radiosensitivity of the brain determined as locomotor activity varied among three strains.ESTRO2

Comet assay, a possible screening assay to classify subgroups of individuals with different radiosensitivity using high throughput scanning system for multiple samples of human blood lymphocytes

This research was designed to identify the correlation between clinical radiosensitivity among breast cancer patients and in vitro radiosensitivity measured by alkaline comet assay in high throughput fashion. In 62 patients with breast cancer and age-matched 41 healthy volunteers, acute adverse effects on skin after radiotherapy were clinically scored according to RTOG grading system. Maximum score during 6 months after radiotherapy was grade 0 for 9 patients, grade 1 for 25 patients, grade 2 for 24 patients, and grade 3 for 4 patients. The parameters of alkaline comet assay were initial damage, which was mean tail moment (MTM) values in irradiated cells in vitro immediately after irradiation with 5 Gy, and % residual damage (RD) at 15 min after irradiation. Correlation between initial damage and skin reaction was found in breast cancer patients with grade 1 and grade 2, 3 (p=0.017). There was no correlation between RD and skin reaction (p=0.056), while large inter-individual variation of RD was revealed among breast cancer patients with grade 0 (17.04 +13.31 %), or grade 2, 3 (16.86 +11.09 %). By introducing a new analyzer, throughput of the comet assay data was highly improved. Our data suggests that the comet assay might be one of supportive assays to classify subpopulation of patients who have different radiosensitivity from normal responders with a fair-poor discriminating capacity of the test to identify the patients with higher risk of developing a severe acute reaction.The 12th International Congress of Radiation Research(ICRR

Microarray Analysis Of The Transcriptional Response To Carbon Ion Irradiation In Murine Tumors

Purpose/Objective(s):The purpose of this study was to identify molecular mechanism induced by carbon ion radiotherapy in order to provide information on potential targets for prediction of its effectiveness.Materials/Methods:Murine squamous cell carcinomas, NR-S1 (resistant to gamma-irradiation), and SCCVII (sensitive), were transplanted in hind legs of C3H/He male mice and established solid tumors (7.5-8.5 mm in diameter) were locally irradiated with carbon ion beam at 30Gy. Carbon-12 ions were accelerated by the Heavy Ion Medical Accelerator in Chiba or HIMAC synchrotron up to 290 MeV/u with a dose rate of approximately 3 Gy /min. Tumor growth delay (TGD) time, reduction rate of tumor, and recurrence rate of tumor were investigated as parameters of radiosensitivity of tumors. The mice were sacrificed and immediately dissected before irradiation and after different time points, such as 6,12,18h, 1, 3, 5, 7, 10, 15, 20 days after irradiation or recurrence for transcriptome assays and pathological investigation. Expression analyses were performed using single-color analysis microarrays consisting of 55k genes. Principal Compornent Analysis (PCA) was used to investigate similarity of comprehensive overview of the changes in gene expression between expression profiles of two tumors. Analysis of variance (ANOVA) was applied to the intensity of each tumor at different time point to evaluate significant differences.Results:TGD time of NR-S1 and SCCVII was 30 days and 56 days, reduction rate of NR-S1 and SCCVII was 40% and 100%, and recurrence rate of NR-S1 and SCCVII was 75% and 50%, respectively.PCA showed that all expression profiles of NR-S1 were identified as a group, while those of SCCVII were identified as another group. Recurred tumors showed different profiles from non-irradiation control tumors. We detected genes, whose expressions were significantly up-regulated or down regulated at each time point after carbon-irradiation (p value< 0.0001). At 6 hours after irradiation, fourteen genes, which were related with cell cycle regulation, were differentially expressed in both tumors. Eleven genes, which were related with inflammation or extracellular matrix, were up-regulated at 6 hours in both tumors, however, their expression changes on time-course were different. Pathological specimen showed duplet cells in both tumors 1 day after irradiation and continuous infiltration of inflammatory cells in SCCVII.Conclusions:Tumor growth assays revealed that two murine tumors, which have different radiosensitivity to gamma irradiation, kept their intrinsic radiosensitivity to carbon-ion irradiation. Transcriptional profiling of two tumors identified a number of carbon-ion irradiation response genes in murine tumors. We have also identified genes as being candidates for predictive markers of radiosensitivity to carbon-ion therapy.48th Annual Meeting of the American Society for Therapeutic Radiology and Oncolog